A fundamental function of epithelia and endothelia is to separate different compartments within the organism and to regulate the exchange of substances between them. The tight junction (TJ) constitutes the barrier both to the passage of ions and molecules through the paracellular pathway and to the movement of proteins and lipids between the apical and the basolateral domains of the plasma membrane. In recent years more than 40 different proteins have been discovered to be located at the TJs of epithelia, endothelia and myelinated cells. This unprecedented expansion of information has changed our view of TJs from merely a paracellular barrier to a complex structure involved in signaling cascades that control cell growth and differentiation. Both cortical and transmembrane proteins integrate TJs. Among the former are scaffolding proteins containing PDZ domains, tumor suppressors, transcription factors and proteins involved in vesicle transport. To date two components of the TJ filaments have been identified: occludin and claudin. The latter is a protein family with more than 20 members. Both occludin and claudins are integral proteins capable of interacting adhesively with complementary molecules on adjacent cells and of co-polymerizing laterally. These advancements in the knowledge of the molecular structure of TJ support previous physiological models that exhibited TJ as dynamic structures that present distinct permeability and morphological characteristics in different tissues and in response to changing natural, pathological or experimental conditions.

Tight junction proteins.

Evaluation of the human genome suggests that all members of the connexin family of gap-junction proteins have now been successfully identified. This large and diverse family of proteins facilitates a number of vital cellular functions coupled with their roles, which range from the intercellular propagation of electrical signals to the selective intercellular passage of small regulatory molecules. Importantly, the extent of gap-junctional intercellular communication is under the direct control of regulatory events associated with channel assembly and turnover, as the vast majority of connexins have remarkably short half-lives of only a few hours. Since most cell types express multiple members of the connexin family, compensatory mechanisms exist to salvage tissue function in cases when one connexin is mutated or lost. However, numerous studies of the last decade have revealed that mutations in connexin genes can also lead to severe and debilitating diseases. In many cases, single point mutations lead to dramatic effects on connexin trafficking, assembly and channel function. This review will assess the current understanding of wild-type and selected disease-linked mutant connexin transport through the secretory pathway, gap-junction assembly at the cell surface, internalization and degradation.

Life cycle of connexins in health and disease

http://csmres.co.uk/cs.public.upd/article-downloads/Future-directions-for-peptide-therapeutics-development.pdf

Future directions for peptide therapeutics development.

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease.

/pdf/remodelling-of-gap-junctions-and-connexin-expression-in-11lt5r0bgt.pdf

Remodelling of gap junctions and connexin expression in diseased myocardium

Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied. Structural proteins, notably zona occludens-1 (ZO-1) and microtubules, have been found recently at gap junctions. Along with the expansion of the list of connexin-associating proteins, reports have appeared that suggest that connexins might have additional roles in addition to their channel function, such as transcriptional and cytoskeletal regulation. Here, gap junction interacting proteins are reviewed and their function is addressed. The striking similarity of proteins present at the cytoplasmic face of tight junctions, adherens junctions and gap junctions and their possible role in gene transcription and cytoskeletal anchorage is highlighted.

/pdf/gap-junctions-and-connexin-interacting-proteins-2hujkvc76u.pdf

Gap junctions and connexin-interacting proteins

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.

Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion.

The intercellular geometry of connexin43 (Cx43) gap junctional coupling is key to coordinated spread of electrical activation through the ventricle of the mammalian heart. A progressive redistribution of electrical and mechanical junctions into intercalated discs occurs during postnatal development. Breakdown of disc-localized pattern in the adult heart, to recapitulate immature distributions, is thought to be key to the genesis of conduction disturbance and arrhythmia. Recently, ZO-1 (a PDZ-MAGUK protein), has been suggested to have a role in generating coupling geometries between myocytes. We therefore investigated the codistribution of ZO-1 with Cx43 and N-cadherin in the adult rat ventricle using quantitative immunoconfocal and immunoelectron microscopy. These analyses indicated that, whereas ZO-1 and Cx43 codistribute within discs, only low to moderate point-by-point colocalization of Cx43 and ZO-1 is found within these domains compared with the relatively high level of colocalization between N-cadherin and ZO-1. By contrast, levels of association between Cx43 and ZO-1 increased rapidly and significantly (P<0.001) after partial or complete enzymatic dissociation of myocytes from intact ventricle--a treatment known to induce gap junction endocytosis. Coimmunoprecipitation using Cx43- and ZO-1-specific antibodies confirmed that significantly (P<0.03) increased ZO-1 is precipitated relative to Cx43 in freshly dissociated myocytes as compared with intact ventricle. On immunoblots, decreases in Cx43 relative mobility, consistent with increased phosphorylation, were observed following myocyte dissociation. The increased ZO-1-Cx43 association that occurs after remodeling of myocyte intercellular contacts indicates the possibility of unanticipated roles for ZO-1 in gap junction turnover during cardiac development and disease processes.

Increased association of ZO-1 with connexin43 during remodeling of cardiac gap junctions.

Intercellular connectivity mediated by gap junctions (GJs) composed of connexin43 (Cx43) is critical to the function of excitable tissues such as the heart and brain. Disruptions to Cx43 GJ organization are thought to be a factor in cardiac arrhythmias and are also implicated in epilepsy. This article is based on a presentation to the 4th Larry and Horti Fairberg Workshop on Interactive and Integrative Cardiology and summarizes the work of Gourdie and his lab on Cx43 GJs in the heart. Background and perspective of recently published studies on the function of Cx43-interacting protein zonula occludens-(ZO)-1 in determining the organization of GJ plaques are provided. In addition how a peptide containing a PDZ-binding sequence of Cx43, developed as part of the work on cardiac GJ organization is also described, which has led to evidence for novel and unexpected roles for Cx43 in modulating healing following tissue injury.

The Unstoppable Connexin43 Carboxyl-Terminus New Roles in Gap Junction Organization and Wound Healing

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.

ZO-1 determines adherens and gap junction localization at intercalated disks

The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.

Ralph J. Barker

Papers

Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion.

Increased association of ZO-1 with connexin43 during remodeling of cardiac gap junctions.

The Unstoppable Connexin43 Carboxyl-Terminus New Roles in Gap Junction Organization and Wound Healing

ZO-1 determines adherens and gap junction localization at intercalated disks

Quantitative analysis of ZO-1 colocalization with Cx43 gap junction plaques in cultures of rat neonatal cardiomyocytes.